Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
  • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
  • C07D409/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/06—Antiasthmatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/08—Antiallergic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

• This invention is directed to a substituted arylmethylamine compound, its preparation, a pharmaceutical composition comprising the compound, its use, and intermediates thereof.
• Mast cell mediated inflammatory conditions are a growing public health* concern. Asthma is frequently characterized by progressive development of hyper-responsiveness of the trachea and bronchi to both immunospecific allergens and generalized chemical or physical stimuLi, which lead to the onset of chronic inflammation.
• Leukocytes containing IgE receptors notably mast cells and basophils, are present in the epithelium and underlying smooth muscle tissues of bronchi. These leukocytes initially become activated by the binding of specific inhaled antigens to the IgE receptors and then release a number of chemical mediators. For example, degranulation of mast cells leads to the release of proteoglycans, peroxidase, arylsulfatase B, chymase, and tryptase, which results in bronchiole constriction.
• Tryptase is stored in the mast cell secretory granules and is the major secretory protease of human mast cells. Tryptase has been implicated in a variety of biological processes, including degradation of vasodilating and bronchorelaxing neuropeptides (Caughey, et al., J. Pharmacol. Exp. Ther., 1988, 244, pages 133-137; Franconi, et al., J. Pharmacol. Exp. Ther., 1988, 248, pages 947-951 ; and Tarn, et al., Am. J. Respir. Cell Mol. Biol., 1990, 3, pages 27-32) and modulation of bronchial responsiveness to histamine (Sekizawa, et al., J. Clin. Invest., 1989, 83, pages 175-179).
• tryptase inhibitors may be useful as anti-inflammatory agents (K Rice, P.A. Sprengler, Current Opinion in Drug Discovery and Development, 1999, 2(5), pages 463-474) particularly in the treatment of chronic asthma (M.Q. Zhang, H. Timmerman, Mediators Inflamm., 1997, 112, pages 311-317), and may also be useful in treating or preventing allergic rhinitis (S. J. Wilson et al, Clin. Exp. Allergy, 1998, 28, pages 220-227), inflammatory bowel disease (S.C. Bischoff et al, Histopathology, 1996, 28, pages 1-13), psoriasis (A. Naukkarinen et al, Arch. Dermatol.
• tryptase has been shown to be a potent mitogen for fibroblasts, suggesting its involvement in the pulmonary fibrosis in asthma and interstitial lung diseases (Ruoss et al., J. Clin. Invest, 1991, 88, pages 493-499).
• tryptase inhibitors may be useful in treating or preventing fibrotic conditions (J.A. Cairns and A.F. Walls, J. Clin. Invest., 1997, 99, pages 1313-1321) for example, fibrosis, sceleroderma, pulmonary fibrosis, liver cirrhosis, myocardial fibrosis, neurofibromas and hypertrophic scars.
• tryptase inhibitors may be useful in treating or preventing myocardial infarction, stroke, angina and other consequences of atherosclerotic plaque rupture (M. Jeziorska et al, J. Pathol., 1997, 182, pages 115-122).
• Tryptase has also been discovered to activate prostromelysin that in turn activates collagenase, thereby initiating the destruction of cartilage and periodontal connective tissue, respectively.
• tryptase inhibitors could be useful in the treatment or prevention of arthritis, periodontal disease, diabetic retinopathy, and tumor growth (W.J. Beil et al, Exp. Hematol., (1998) 26, pages 158-169). Also, tryptase inhibitors may be useful in the treatment of anaphylaxis (L.B. Schwarz et al, J. Clin. Invest., 1995, 96, pages 2702-2710), multiple sclerosis (M. Steinhoff et al, Nat. Med. (N. Y.), 2000, 6(2), pages 151-158), peptic ulcers and syncytial viral infections.
• the present invention is directed to a pharmaceutical composition
• a pharmaceutical composition comprising a pharmaceutically effective amount of the compound of formula I, and a pharmaceutically acceptable carrier.
• the present invention is directed to the use of a compound of formula I as an inhibitor of tryptase, comprising introducing the compound into a composition comprising tryptase.
• the present invention is directed to the use of a compound of formula I for treating a patient suffering from, or subject to, a physiological condition in need of amelioration of an inhibitor of tryptase comprising administering to the patient a therapeutically effective amount of the compound of Claim 1
• Figure I Compound I levels in plasma, bronchoalveolar lavage (BAL) fluid and lung, measured 2 hour after dosing compound 1 at 1 mg/kg, p.o. Values are mean ⁇ SE of 6-8 animals.
• FIG. 1 Plasma and lung compound I levels measured 24 hour post dosing. Values are mean ⁇ SE of 3-4 animals.
• the term "compound of the present invention”, and equivalent expressions, are meant to embrace the compound of formula I, as hereinbefore described, which expression includes the prodrug, the pharmaceutically acceptable salt and the solvate, e.g., hydrate.
• reference to intermediates, whether or not they themselves are claimed, is meant to embrace the salts, and solvates, where the context so permits.
• particular instances when the context so permits are sometimes indicated in the text, but these instances are purely illustrative and they are not intended to exclude other instances when the context so permits.
• treatment includes prophylactic therapy as well as treatment of an established condition.
• Patient means a human or other mammal.
• Effective amount is meant to describe an amount of a compound effective in producing the desired therapeutic effect.
• Prodrug means a compound that is suitable for adrninistration to a patient without undue toxicity, irritation, allergic response, and the like, and is convertible in vivo by metabolic means (e.g. by hydrolysis) to the compound of the present invention.
• metabolic means e.g. by hydrolysis
• the present invention is directed to the use of the compound of formula I for treating a patient suffering from a physiological condition that can be ameliorated by administering to the patient a therapeutically effective amount of the compound of formula I.
• physiological conditions include, but certainly are not limited to inflammatory diseases, e.g., joint inflammation, arthritis, rheumatoid arthritis, rheumatoid spondylitis, gouty arthritis, traumatic arthritis, rubella arthritis, psoriatic arthritis, and other chronic inflammatory joint diseases.
• physiological conditions include physiological conditions such as chronic obstructive pulmonary disease (COPD), COPD exacerbations, joint cartilage destruction,ocular conjunctivitis, vernal conjunctivitis, inflammatory bowel disease, asthma, allergic rhinitis, interstitial lung diseases, fibrosis, sceleroderma, pulmonary fibrosis, liver cirrhosis, myocardial fibrosis, neurofibromas, hypertrophic scars, various dermatological conditions, for example, atopic dermatitis and psoriasis, myocardial infarction, stroke, angina and other consequences of atherosclerotic plaque rupture, as well as periodontal disease, diabetic retinopathy, tumor growth, anaphylaxis, multiple sclerosis, peptic ulcers, and syncytial viral infections.
• COPD chronic obstructive pulmonary disease
• COPD chronic obstructive pulmonary disease
• COPD chronic obstructive pulmonary disease
• the present invention is directed to the use of a compound of formula I for treating a patient suffering from asthma, comprising administering to the patient a physiologically effective amount of the compound.
• the present invention is directed to the use of a compound of formula I for treating a patient suffering from COPD, comprising administering to the patient a physiologically effective amount of the compound.
• the present invention is directed to the use of a compound of formula I for treating a patient suffering from COPD exacerbations, comprising administering to the patient a physiologically effective amount of the compound.
• the present invention is directed to the use of a compound of formula I for treating a patient suffering from allergic rhinitis, comprising administering to the patient a physiologically effective amount of the compound.
• the present invention is directed to the use of a compound of formula I for treating a patient suffering from joint inflammation, comprising administering to the patient a physiologically effective amount of the compound.
• the present invention is directed to the use of a compound of formula I for treating a patient suffering from inflammatory bowel disease, comprising administering to the patient a physiologically effective amount of the compound.
• the present invention extends to a pharmaceutical composition
• a pharmaceutical composition comprising the compound of formula I, a second compound selected from the group consisting of a beta andrenergic agonist, an anticholinergic, an anti-inflammatory corticosteroid, and an anti-inflammatory agent, and a pharmaceutically acceptable carrier thereof, hi such a composition the compound of formula I and the second compound are present in amounts such that provide a therapeutically efficacious activity, i.e., additive or synergistic effect.
• a pharmaceutical composition comprising the compound of formula I, a second compound selected from the group consisting of a beta andrenergic agonist, an anticholinergic, an anti-inflammatory corticosteroid, and an anti-inflammatory agent, and a pharmaceutically acceptable carrier thereof, hi such a composition the compound of formula I and the second compound are present in amounts such that provide a therapeutically efficacious activity, i.e., additive or synergistic effect.
• Particular inflammatory diseases or disorders include, but is not limited to, asthma.
• the present invention is directed to a method for treating a patient suffering from an inflammatory disorder, comprising administering to the patient the compound of formula I and a second compound selected from the group consisting of a beta andrenergic agonist, an anticholinergic, an anti-inflammatory corticosteroid, and an anti-inflammatory agent.
• the compound of formula I and the second compound are present in amounts such that provide a therapeutically efficacious activity, i.e., additive or synergistic effect.
• the compound of the present invention can be administered to the patient before a second compound, a second compound can be administered to the patient before a compound of the present invention, or a compound of the present invention and a second compound can be administered concurrently.
• Particular examples of andrenergic agonists, anticholinergics, anti-inflammatory corticosteroids, and anti-inflammatory agents having application according to the method are described infra.
• the compound of the present invention exhibits useful pharmacological activity and accordingly may be incorporated into a pharmaceutical composition and used in the treatment of patients suffering from certain medical disorders.
• the present invention thus provides, according to a further aspect, pharmaceutical compositions comprising the compound of the invention, and a pharmaceutically acceptable carrier thereof.
• pharmaceutically acceptable preferably means approved by a regulatory agency of a government, in particular the Federal government or a state government, or listed in the U.S. Pharmacopoeia or another generally recognized pharmacopoeia for use in animals, and more particularly in humans. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.
• compositions according to the present invention can be prepared according to the customary methods, using one or more pharmaceutically acceptable adjuvants or excipients.
• the adjuvants comprise, inter alia, diluents, fillers, binders, disintegrants, glidants, lubricants, surfactants, sterile aqueous media and the various non-toxic organic solvents.
• the compositions may be presented in the form of tablets, capsules, pills, sustained release formulations, granules, powders, aqueous solutions or suspensions, injectable solutions, elixirs or syrups, and can contain one or more agents chosen from the group comprising sweeteners, flavorings, colorings, or stabilizers in order to obtain pharmaceutically acceptable preparations.
• excipients such as lactose, microcrystalline cellulose, pregelatinized starch, unmodified starch, silicif ⁇ ed microcrystalline cellulose, mannitol, sorbitol, xylitol, dextrates, fructose, sodium citrate, calcium carbonate, dicalcium phosphate dihydrate, anhydrous dicalcium phosphate, calcium sulfate, along with binders such as polyvinylpyrollidone, hydroxypropylmethyl cellulose, ethyl cellulose, hydroxyethyl cellulose, methyl cellulose, sodium carboxymethyl cellulose, pregelatinized starch, starch, polyethylene glycols, polyethylene oxide, polycarbophils, gelatin and acacia and disintegrating agents such as sodium croscarmellose, sodium
• fillers such as lactose, microcrystalline cellulose, pregelatinized starch, unmodified starch, silicified microcrystalline cellulose alone or a mixture of two or more fillers, with and without binders as described above along with suitable wetting agent (s), disintegrants, glidants, lubricants, etc. as listed above.
• suitable wetting agent s
• disintegrants glidants, lubricants, etc.
• aqueous suspensions they can contain emulsifying agents or agents which facilitate suspension.
• Diluents such as sucrose, ethanol, polyethylene glycol, propylene glycol, glycerol and chloroform or mixtures thereof may also be used.
• Such pharmaceutically acceptable carriers can also be sterile water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
• Suitable pharmaceutical excipients include mannitol, human serum albumin (HSA), starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium carbonate, magnesium stearate, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
• HSA human serum albumin
• starch glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium carbonate, magnesium stearate, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
• a pharmaceutical composition of the present invention will contain a therapeutically effective amount of the active compound together with a suitable amount of carrier so as to provide the form for proper administration to the patient. While intravenous injection is a very effective form of administration, other modes can be employed, such as by injection, or by oral, nasal or parenteral administration, which are discussed infra. Methods of Treatment
• the compound of formula I possesses tryptase inhibition activity according to tests described in the literature and described hereinafter, and which test results are believed to correlate to pharmacological activity in humans and other mammals.
• the present invention is directed to the use of formula I or a composition comprising it for treating a patient suffering from, or subject to, a condition that can be ameliorated by the administration of an inhibitor of tryptase.
• the compound of formula I is useful for treating an inflammatory disease, for example, joint inflammation, including arthritis, rheumatoid arthritis and other arthritic condition such as rheumatoid spondylitis, gouty arthritis, traumatic arthritis, rubella arthritis, psoriatic arthritis, osteoarthritis or other chronic inflammatory joint disease, or diseases of joint cartilage destruction, Cular conjunctivitis, vernal conjunctivitis, inflammatory bowel disease, asthma, allergic rhinitis, interstitial lung diseases, fibrosis, sceleroderma, pulmonary fibrosis, liver cirrhosis, myocardial fibrosis, neurofibromas, hypertrophic scars, various dermatological conditions, for example, atopic dermatitis and psoriasis, myocardial infarction, stroke, angina or other consequences of atherosclerotic plaque rupture, as well as periodontal disease, diabetic retinopathy, tumor growth, anaphyl
• A-ccording to a further feature of the invention there is provided a method for the treatment of a human or animal patient suffering from, or subject to, conditions which can be ameliorated by the administration of an inhibitor of tryptase, for example conditions as hereinbefore described, which comprises the administration to the patient of an effective amount of compound of the invention or a composition containing a compound of the invention.
• beta-adrenergic agonists such as albuterol, terbutaline, formoterol, fenoterol or prenaline can be included, as can anticholinergics such as ipratropium bromide, anti-inflarnmatory corticosteroids such as beclomethasone dipropionate, triamcinolone acetonide, flunisolide or dexametlxasone, and anti-inflammatory agents such as sodium cromoglycate and nedocromil sodium.
• beta-adrenergic agonists such as albuterol, terbutaline, formoterol, fenoterol or prenaline
• anticholinergics such as ipratropium bromide
• anti-inflarnmatory corticosteroids such as beclomethasone dipropionate, triamcinolone acetonide, flunisolide or dexametlxasone
• anti-inflammatory agents such as sodium
• the present invention extends to a pharmaceutical composition
• a pharmaceutical composition comprising the compound of formula I and a second compound selected from the group consisting of a beta andrenergic agonist, an anticholinergic, an anti-inflammatory corticosteroid, and an anti-inflammatory agent; and a pharmaceutically acceptable carrier thereof.
• a pharmaceutical composition comprising the compound of formula I and a second compound selected from the group consisting of a beta andrenergic agonist, an anticholinergic, an anti-inflammatory corticosteroid, and an anti-inflammatory agent; and a pharmaceutically acceptable carrier thereof.
• Particular pharmaceutical carriers having applications in this pharmaceutical composition are described herein.
• the present invention extends to a method for treating a patient suffering from asthma, comprising administering the patient the compound of the present invention, and a second compound selected from the group consisting of a beta andrenergic agonist, an anticholinergic, an anti- inflammatory corticosteroid, and an anti-inflammatory agent.
• a second compound selected from the group consisting of a beta andrenergic agonist, an anticholinergic, an anti- inflammatory corticosteroid, and an anti-inflammatory agent.
• the compound of the present invention can be administered prior to the administration of the second compound, the compound of the present invention can be administered after administration of the second compound, or the compound of the present invention and the second compound can be administered concurrently. Modes of Delivery.
• the compound of formula I, or a pharmaceutical composition comprising the compound may be introduced parenterally, transmucosally, e.g., orally, nasally, pulmonarily, or rectally, or transdermally to a patient.
• Oral Delivery e.g., orally, nasally, pulmonarily, or rectally, or transdermally to a patient.
• Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets or pellets.
• liposomal or proteinoid encapsulation may be used to formulate the present compositions (as, for example, proteinoid microspheres reported in U.S. Patent No. 4,925,673).
• Liposomal encapsulation may be used and the liposomes may be derivatized with various polymers (e.g., U.S. Patent No. 5,013,556).
• the formulation will include a compound of the present invention, and inert ingredients that allow for protection against the stomach environment, and release of the biologically active material, i.e., a compound of the present invention, in the intestine.
• oral dosage forms of the compound of the present invention are also contemplated.
• Such a compound may be chemically modified so that oral delivery is more efficacious.
• the chemical modification contemplated is the attachment of at least one moiety to the component molecule itself, where said moiety permits (a) inhibition of proteolysis; and (b) uptake into the blood stream from the stomach or intestine.
• moieties include: polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline.
• the location of release may be the stomach, the small intestine (the duodenum, the jejunum, or the ileum), or the large intestine.
• the stomach the small intestine (the duodenum, the jejunum, or the ileum), or the large intestine.
• One skilled in the art has available formulations that will not dissolve in the stomach, yet will release the material in the duodenum or elsewhere in the intestine.
• the release will avoid the deleterious effects of the stomach environment, either by protection of the compound of the present invention, or by release of the compound beyond the stomach environment, such as in the intestine.
• a coating impermeable to at least pH 5.0 is essential.
• cellulose acetate trimellitate hydroxypropylmethylcellulose phthalate
• HPMCP 50 HPMCP 55
• PVAP polyvinyl acetate phthalate
• Eudragit L30D Aquateric
• CAP cellulose acetate phthalate
• Eudragit S Eudragit S, and shellac.
• a coating or mixture of coatings can also be used on tablets, which are not intended for protection against the stomach. This can include sugar coatings, or coatings that make the tablet easier to swallow.
• Capsules may consist of a hard shell (such as gelatin) for delivery of dry therapeutic i.e. powder; for liquid forms, a soft gelatin shell may be used.
• the shell material of cachets could be thick starch or other edible paper. For pills, lozenges, molded tablets or tablet triturates, moist massing techniques can be used.
• the therapeutic can be included in the formulation as fine multi-particulates in the form of granules or pellets of particle size about 1 mm.
• the formulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets.
• the therapeutic could be prepared by compression.
• Colorants and flavoring agents may all be included.
• the compound of the present invention may be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents.
• diluents could include carbohydrates, especially mannitol, ⁇ -lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch.
• Certain inorganic salts may be also be used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride.
• Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.
• Disintegrants may be included in the formulation of the therapeutic into a solid dosage form.
• Materials used as disintegrates include, but are not limited to starch, including the commercial disintegrant based on starch, Explotab.
• Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite may all be used.
• Another form of the disintegrants are the insoluble cationic exchange resins.
• Powdered gums may be used as disintegrants and as binders and these can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.
• Binders may be used to hold the therapeutic agent together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both be used in alcoholic solutions to granulate the therapeutic.
• MC methyl cellulose
• EC ethyl cellulose
• CMC carboxymethyl cellulose
• PVP polyvinyl pyrrolidone
• HPMC hydroxypropylmethyl cellulose
• Lubricants may fee used as a layer between the therapeutic and the die wall, and these can include but are not limited to; stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000.
• the glidants may include starch, talc, pyrogenic silica and hydrated silicoaluminate.
• surfactant might be added as a wetting agent.
• Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate.
• anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate.
• Cationic detergents might be used and could include benzalkonium chloride or benzethomium chloride.
• non-ionic detergents that could be included in the formulation as surfactants are lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the formulation of a compound of the present invention either alone or as a mixture in different ratios.
• Additives that potentially enhance uptake of the compound of the present invention are, for instance, the fatty acids oleic acid, linoleic acid and linolenic acid.
• Controlled release oral formulation may be desirable.
• the drug could be incorporated into ant inert matrix that permits release by either diffusion or leaching mechanisms, e.g., gums. Slowly degenerating matrices may also be incorporated into the formulation.
• Some enteric coatings also have a delayed release effect.
• Another form of a controlled release of this therapeutic is by a method based on the Oros therapeutic system (Alza Corp.), i.e. the drug is enclosed in a semipermeable membrane which allows water to enter and push drug out through a single small opening d e to osmotic effects.
• coatings may be used for the formulation. These include a variety of sugars that could be applied in a coating pan.
• the therapeutic agent could also be given in a film-coated tablet and the materials used in this instance are divided into 2 groups.
• the first are the non-enteric materials and include methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose, providone and the polyethylene glycols.
• the second groixp consists of the enteric materials that are commonly esters of phthalic acid. A mix of materials might be used to provide the optimum film coating. Film coating may be carried out in a pan-coater or in a fluidized bed or by cor ⁇ pression coating. Pulmonary Delivery
• pulmonary delivery of the compound of the present invention is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream.
• Other reports of this include Adjei et al., 1990, Pharmaceutical -Research, 7:565-569; Adjei et al., 1990, International Journal of Pharmaceutics, 63:135-144 (leup_rolide acetate); Braquet et al., 1989, Journal of Cardiovascular Pharmacology, 13(suppl. 5): 143-146 (endothelin-1); Hubbard et al., 1989, Annals of internal Medicine, Vol.
• Contemplated for use in the practice of this invention are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art.
• Some specific examples of commercially available devices suitable for the practice of this invention are the Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Missouri; the Acorn II nebulizer, manufactured by Marquest Medical Products, Englewood, Colorado; the Ventolin metered dose inhaler, manufactured by Glaxo Inc., Resea-rch Triangle Park, North Carolina; and the Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford, Massachusetts, to name only a few. All such devices require the use of formulations suitable for the dispensing of the compound of the present invention.
• each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to the usual diluents, adjuvants and/or carriers useful in therapy. Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated.
• a chemically modified compound of the present invention may also be prepared in different formulations depending on the type of chemical modification or the type of device employed.
• Formulations suitable for use with a nebulizer will typically comprise the compound of the present invention dissolved in water at a concentration of about 0.1 to 25 mg of compound per mL of solution.
• the formulation may also include a buffer and a simple sugar (e.g., for stabilization and regulation of osmotic pressure).
• the netoulizer formulation may also contain a surfactant, to reduce or prevent surface induced aggregation of the compound caused by atomization of the solution in forming the aerosol.
• Formulations for use with a metered-dose inhaler device will generally comprise a finely divided powder containing the compound of the invention suspended in a propellant with the aid of a surfactant.
• the propellant may be any conventional material employed for this purpose, such as a chlorofluorocarbon, hydrochlorofluorocarbon, hydrofluorocarbon, or hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or combinations thereof.
• Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid may also be useful as a surfactant.
• Formulations for dispensing from a powder inhaler device ⁇ vill comprise a finely divided dry powder containing the compound of the invention, and may also include a bulking agent, such as lactose, sorbitol, sucrose, or mannitol in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation.
• the compound of the present invention should most advantageously be prepared in particulate form with an average particle size of less than 10 mm (or microns), most preferably 0.5 to 5 mm, for most effective delivery to the distal lung. Nasal Delivery
• Nasal delivery of the compound of the present invention is also contemplated. Nasal delivery allows the passage of the compound to the blood stream directly after administering the therapeutic product to the nose, without the necessity for deposition of the product in the lung.
• Formulations for nasal delivery include those with dextran or cyclodextran.
• Transdermal patches are described in for example, U.S. Patent No. 5,407,713, issued April L 8, 1995 to Rolando et al.; U.S. Patent No. 5,352,456, issued October 4, 1994 to Fallon et al.; U.S. Patent No. 5,332,213 issued August 9, 1994 to D'Angelo et al.; U.S. Patent No. 5,336,168, issued August 9, 1994 to Sibalis; U.S. Patent No. 5,290,561, issued March 1, 1994 to Farhadieh et al.; U.S. Patent No.
• a transdermal route of adn ⁇ inistration may be enhanced by use of a dermal penetration enhancer, e.g., such as enhancers descri " bed in U.S. Patent No. 5,164,189 supra), U.S. Patent No. 5,008,110 (supra), and U.S. Patent No. 4,879,119, issued November 7, 1989 to Aruga et al, the disclosure of each of which is incorporated herein by reference in its entirety.
• a dermal penetration enhancer e.g., such as enhancers descri " bed in U.S. Patent No. 5,164,189 supra
• U.S. Patent No. 5,008,110 supra
• U.S. Patent No. 4,879,119 issued November 7, 1989 to Aruga et al, the disclosure of each of which is incorporated herein by reference in its entirety.
• gels water or alcohol based
• creams or ointments containing compounds of the invention may be used.
• Compounds of the invention may also be incorporated in a gel or matrix base for application in a patch, which would allow a controlled release of compound through the transdermal barrier. Rectal Administration
• Solid compositions for rectal administration include suppositories formulated in accordance with known methods and containing the compound of the invention.
• the percentage of active ingredient in the composition of the invention may be varied, it being necessary that it should constitute a proportion such that a suitable dosage shall be obtained. Obviously, several unit dosage forms may be administered at about the s me time. The dose employed will be determined by the physician, and depends upon the desired therapeutic effect, the route of administration and the duration of the treatment, and the condition of the patient.
• the doses are generally from about 0.001 to about 50, preferably about 0.001 to about 5, mg/kg body weight per day by inhalation, from about 0.01 to about 100, preferably 0.1 to 70, more especially 0.5 to 10, mg/kg body weight per day by oral administration, and from about 0.001 to about 10, preferably 0.01 to 1, mg/kg body weight per day by intravenous administration, i each particular case, the doses will be determined in accordance with the factors distinctive to the subject to be treated, such as age, weight, general state of health and other characteristics which can influence the efficacy of the medicinal product.
• the compound according to the invention may be administered as frequently as necessary in order to obtain the desired therapeutic effect. Some patients-; may respond rapidly to a higher or lower dose and may find much weaker maintenance doses adequate. For other patients, it may be necessary to have long-term treatments at the rate of 1 to 4 doses per day, in accordance with the physiological requirements of each particular patient. Generally, the active product may be administered orally 1 to 4 times per day. Of course, for some patients, it will be necessary to prescribe not more than one or two doses per day.
• a patient in whom administration of the compound of the present invention is an effective therapeutic regimen is preferably a human, but can be any animal.
• the methods and pharmaceutical compositions of the present invention are particularly suited to administration to any animal, particularly a mammal, and including, but by no means limited to, domestic animals, such as feline or canine subjects, farm animals, such as but not limited to bovine, equine, caprine, ovine, and porcine subjects, wild animals (whether in the wild or in a zoological garden), research animals, such as mice, rats, rabbits, goats, sheep, pigs, dogs, cats, etc., avian species, such as chickens, turkeys, songbirds, etc., i.e., for veterinary medical use.
• domestic animals such as feline or canine subjects
• farm animals such as but not limited to bovine, equine, caprine, ovine, and porcine subjects
• wild animals whether in the wild or in a zoological garden
• research animals such as mice,
• the compound of formula I may be prepared by the application or adaptation of known methods, by which is meant methods used heretofore or described in the literature, for example those described by R.C.Larock in Comprehensive Organic Transformations, VCH publishers, 1989, or as described herein.
• the compound of the present invention is an achiral compound whose preparation is comprised of a convergent synthesis.
• Scheme I shows the procedures that culminate in the preparation of amine 10.
• Scheme II shows the procedures that culminate in the preparation of acid 16.
• Scheme III shows the procedures that culminate in the preparation of the compound of formula I, to yield the compound of formula I in a two-step sequence.
• the preparation of 10, 16 and the compound of formula I of the present invention are discussed in turn below.
• Compound 2 is converted to compound 3 by protecting the amino groixp with an amino protecting agent, such as l,2-bis(chlorodimethylsilyl)ethane, in the presence of a tertiary amine, such as triethylamine, in a suitable inert solvent, such as dichloromethane, to yield protected compound 3.
• an amino protecting agent such as l,2-bis(chlorodimethylsilyl)ethane
• a tertiary amine such as triethylamine
• a suitable inert solvent such as dichloromethane
• Compound 3 is converted to compound 5 by alkylating compound 4 using compound 3 under alkylating conditions comprising a strong base, such as n-butyllithium, in a suitable aprotic solvent, such as tetrahydrofuran, to yield the hydroxyl derivative compound 5.
• a strong base such as n-butyllithium
• a suitable aprotic solvent such as tetrahydrofuran
• Compound 5 is converted to compound 6 by deprotecting the amino group thereof with a deprotecting agent, such as a strong inorganic acid, such as phosphoric acid, in the presence of an inert solvent, such as heptane, to yield deprotected compound 6.
• a deprotecting agent such as a strong inorganic acid, such as phosphoric acid
• an inert solvent such as heptane
• Compound 6 is converted to compound 7 by dehydrating, using an strong inorganic acid, such as phosphoric acid, and subsequent neutralizing the product using a strong inorganic base, such as aqueous sodium hydroxide to yield the dehydrated compound 7.
• an strong inorganic acid such as phosphoric acid
• a strong inorganic base such as aqueous sodium hydroxide
• Compound 7 is converted to compound 8 by protecting the amino gronp with an amino protecting agent, such as boc anhydride, in a mixed aqueous/organic solvent system, wherein the organic solvent is a polar organic solvent such as methanol, using a strong inorganic base, such as aqueous sodium hydroxide to yield boc-protected compound 8.
• Compound 8 is converted to compound 9 by hydrogenating using a reducing agent, such as palladium hydroxide on carbon (20%), in a mixed solvent system, such as methanolic acetic a.cid to yield the deprotected piperidine salt, compound 9.
• an amino protecting agent such as boc anhydride
• Compound 9 is converted to its free base form, compound 10, by neutralizing using a strong inorganic base, such as aqueous sodium hydroxide, to yield final compound 10.
• a strong inorganic base such as aqueous sodium hydroxide
• Carbon disulfide is converted to compound 11 by acylating using the acylating agent propyl chloroformate in the presence of a strong inorganic base, such as potassium hydroxide, in n-propanol to yield compound 11.
• a strong inorganic base such as potassium hydroxide
• Compound 11 is converted to compound 12 by alkylating acetone with compound 11 in the presence of a strong base such as sodium hydride to yield alkylated compound 12.
• Compound 12 is converted to compound 13 by alkylating compound 12 with ⁇ -bromo rxtethyl acetate in the presence of a tertiary amine such as triethylamine to yield the alkylated compound 13.
• Compound 13 is converted to compound 14 by cyclizing under strong base conditions, such as sodium methoxide, in the presence of a protic, organic solvent such as methanol to yield the cyclized compound 14.
• strong base conditions such as sodium methoxide
• a protic, organic solvent such as methanol
• Compound 14 is converted to compound 15 by brominating in an inert organic solvent, such as trichloromethane or a TBME/heptane mixture, to yield the brominated compound 15.
• an inert organic solvent such as trichloromethane or a TBME/heptane mixture
• Compound 15 is converted to compound 16 by hydrolyzing using a strong inorganic base, such as lithium hydroxide.
• Compound 16 is converted to compound 17 by coupling with compound 10 using a coupling agent, such as TPTU/HOBT or EDC, in the presence of an inert solvent, such as dichloromethane, and a tertiary amine such as diisopropyl ethylamine under anhydrous conditions, to yield the coupled compound 17.
• a coupling agent such as TPTU/HOBT or EDC
• an inert solvent such as dichloromethane
• a tertiary amine such as diisopropyl ethylamine under anhydrous conditions
• Compound 17 is converted to compound I by deprotecting under strong acid conditions , such as hydrochloric acid, in the presence of a polar organic solvent, such as dioxane, to yield the deprotected compound I.
• strong acid conditions such as hydrochloric acid
• a polar organic solvent such as dioxane
• the compound of the present invention is basic, and such compound is useful in the form of the free base or in the form of a pharmaceutically acceptable acid addition salt thereof.
• Acid addition salts may be a more convenient form for use; and in practice, use of the salt form inherently amounts to use of the free base form.
• the acids which can be used to prepare the acid addition salts include preferably those which produce, when combined with the free base, pharmaceutically acceptable salts, that is, salts whose anions are non-toxic to the patient in pharmaceutical doses of the salts, so that the beneficial inhibitory effects inherent in the free base are not vitiated by side effects ascribable to the anions.
• compositions of said basic compound are preferred, all acid addition salts are useful as sources of the free base form even if the particular salt, per se, is desired only as an intermediate product as, for example, when the salt is formed only for purposes of purification, and identification, or when it is used as intermediate in preparing a pharmaceutically acceptable salt by ion exchange procedures.
• Pharmaceutically acceptable salts within the scope of the invention include those derived from mineral acids and organic acids, and include hydrohalides, e.g.
• hydrochloride and hydrobromide sulfates, phosphates, nitrates, sulfamates, acetates, citrates, lactates, tartrates, malonates, oxalates, salicylates, propionates, succinates, fumarates, maleates, methylene-bis-b- hydroxynaphthoates, benzoates, tosylates, gentisates, isethionates, di-p-toluoyltartrates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, cyclohexylsulfamates and quinates.
• a more particular salt is salt of the compound of formula I is the hydrochloride salt.
• salts of the compound of the invention are useful for the purposes of purification of the compound, for example by exploitation of the solubility differences between the salts and the parent compound, side products and/or starting materials by techniques well known to those skilled in the art.
• the acid addition salt of the compound of this invention may be prepared by reaction of the free base with the appropriate acid, by the application or adaptation of known methods.
• the acid addition salts of the compound of this invention may be prepared either by dissolving the free base in water or aqueous alcohol solution or other suitable solvents containing the appropriate acid and isolating the salt by evaporating the solution, or by reacting the free base and acid in an organic solvent, in which case the salt separates directly or can be obtained by concentration of the solution.
• the acid addition salts of the compound of this invention can be regenerated from the salts by the application or adaptation of known methods.
• the parent compound of the invention can be regenerated from their acid addition salts by treatment with an alkali, e.g. aqueous sodium bicarbonate solution or aqueous ammonia solution.
• the starting materials and intermediates may be prepared by the application or adaptation of known methods, for example methods as described in the Reference Examples or their obvious chemical equivalents.
• Step 2 Preparation of l-Benzyl-4-[2-fluoro-5-(2,2,5,5-tetramethyl-[l,2,5]-azadisilolidin-l-ylmethyl) phenyl] piperidin-4-ol (Compound 5)
• a solution of compound 3 (159 g, 0.4574 mol) in 1.5 L of anhydrous THF is placed in a 5-L, three-neck, round-bottom flask equipped with N 2 blanket, Teflon-coated thermocouple temperature sensor, and mechanical stirring and is cooled to -75 °C.
• To this stirred solution is added drop-wise a 2.5 M solution of n-butyl lithium (192 ml, 0.48 mol, 1.05 equiv.) over about 1 hour while maintaining the reaction mixture temperature between -72°C to -75°C.
• Step 3 Preparation of 4-(5-Aminomethyl-2-fluorophenyl)-l-benzylpiperidin-4-ol (phosphate salt) (Compound 6)
• the crude compound 5 (-295 g) is diluted with 1 L of methylene chloride and transfened to a 3-L, three-neck, round-bottom flask equipped with Teflon-coated thermocouple temperature sensor, and mechanical stirring. To this solution is slowly added 53 g of 85% H 3 PO (1 equiv.) while stirring (exothermic) and the mixture is concentrated in vacuo (40°C). 1 L of methylene chloride is added and the mixture is concentrated in vacuo (40°C, 1 mbar) to yellow foam, which is then shrrried in 1 L of heptane. The solid is formed and isolated by filtration. The tan solid is dried to give 190 g of the compound 6. MS: m/z 315 (M+H) found.
• Step 4 Preparation of 3-(l-benzyl-l,2,3,6-tetrahydro-pyridin-4-yl)-4-fluoro-benzylamine (Compound 7)
• a total of 190 g of the dried compound 6 is transfened to 2-L, three-neck, round-bottom flask equipped with Teflon-coated thermocouple temperature sensor, and mechanical stirring. A total of 600 mL of 85% H 3 PO 4 is added. The resulting suspension is gradually heated to 100°C while stirring. The reaction is monitored by HPLC for the reaction completion (typically 2 to 3 hours). Once the reaction completed, the reaction solution is cooled to room temperature, and diluted with 800 mL of water. The aqueous layer is washed with ether (2 x 200 mL). The aqueous layer is then neutralized with 50% aq. NaOH to pH > 9 while maintaining temperature below 30 °C.
• This aqueous solution is a very concentrated buffer system in which the pH remains unchanged at around 7-8 till the neutralization point is reached. Confirmation for complete neutralization is effected by adding a few drops of base to a small sample of the supernatant liquid to insure that no additional precipitation is observed. A large amount of salt precipitated out.
• the mixture is filtered and the solid is rinsed with DCM (methylene dichloride) (approximate 3 L) and 2 L of water. The organic layer (bottom) is separated and washed with water (2 x 1 L). The organic solution is concentrated in vacuo (40°C) to give 107 g of brown oil, compound 7. MS: m/z 297 (M+H) found.
• Step 5 Preparation of [3-(l-Benzyl-l,2,3,6-tetrahydropyridin-4-yl)-4-fluoro-benzyl] carbamic acid tert-butyl ester (Compound 8)
• Step 6 Preparation of 6 (4-Fluoro-3-piperidin-4-yl-benzyl) carbamic acid tert-butyl ester, acetate salt (Compound 9)
• Step 7 Preparation of (4-Fluoro-3-piperidin-4-yl-benzyl) carbamic acid tert-butyl ester (Compound 10)
• the acetate salt of compound 9 (51 g) is dissolved in 400 mL of water and the pH is adjusted to 5 with 2N HC1.
• the aqueous solution is washed with ether (2 x 200 mL).
• the aqueous layer is neutralized to pH > 12 with 50% aq. NaOH. and extracted with ether (2 x 300 mL).
• the organic layer is washed with water and dried over Na2SO4, and then concentrated to oil.
• To the oil is added 200 mL of n-pentane and stirred for 3 hours.
• the product solid is isolated by filtration, washed with n-pentane, and dried at room temperature under house vacuum for 24 h to afford 42 g of compound 10 (98%) as a white solid.
• n-pentane and ether up to 1: 1 by volume
• the product is very soluble in ether, so if more ether is used; then more product will be lost, thereby resulting in a lower yield.
• Step 4 Preparation of 3-methyl-5-propoxy-thiophene-2-carboxylic acid methyl ester (compound 14)
• a mixture of 2.61 g (11.24) compound 13 and 2 mL 0.5M sodium methoxide/methanol in 40 mL methanol is heated at 70-73 °C for 40 min.
• the mixture is then poured into ether/ice-water.
• the organic layer is separated and concentrated.
• the crude product is purified by silica gel chromatography, eluting with DCM to obtain 1.85 g (76.8 yield) of compound 14.
• Step 2 Preparation of [4-(5-aminomethyl-2-fluoro-phenyl)-piperidin-l-yl]-(4-bromo-3-methyl-5- propoxy-thiophen-2-yl)-methanone hydrochloride (compound I)
• Tryptase inhibition activity is confirmed using either isolated human lung tryptase or recombinant human ⁇ tryptase expressed in yeast cells. Essentially equivalent results are obtained using isolated native enzyme or the expressed enzyme.
• the assay procedure employs a 96 well microplate (Costar 3590) using L-pyroglutamyl-L-prolyl-L-arginine-p ⁇ r -nitroanilide (S2366: Quadratech) as substrate (essentially as described by McEuen et. al. Biochem Pharm, 1996, 52, pages 331-340). Assays are performed at room temperature using 0.5mM substrate (2 x K m ) and the microplate is read on a microplate reader (Beckman Biomek Plate reader) at 405 nm wavelength.
• Assay buffer 50 mM Tris (pH 8.2), 100 mM NaCl, 0.05% Tween 20, 50 ⁇ g/mL heparin.
• Substrate S2366 (Stock solutions of 2.5 mM).
• Non-specific 60 ⁇ L of substrate, 40 ⁇ L of buffer (No DMSO) Protocol ICsn and K determination)
• the protocol is essentially the same as above except that the compound is added in duplicates at the following final concentrations: 0.01, 0.03, 0.1, 0.3, 1, 3, 10 ⁇ M (All dilutions carried out manually).
• a standard compound is used to derive IC 50 for comparison.
• the ⁇ -Tryptase inhibitory potency for the compound of formula I is IC 50 and Kj values of 76 11M and 15 nM respectively.
• Sensitization and drug treatment Male Hartley guinea pigs (225-250 g) are sensitized with ovalbumin (0.5 mL of 1% solution, i.p. and s.c). On day 4, animals received a booster injection (i.p.) of 0.5 mL of 1% ovalbumin. On day 21, animals are orally dosed (2mL/kg) with either vehicle (0.5% methylcellulose/0.2% Tween 80) or test compound 2 hours prior to antigen challenge. Thirty minutes before antigen challenge the animals are also injected with mepyramine (30 mg/kg, i.p.) to prevent anaphylactic collapse. Animals are then exposed for 5 minutes to an aerosol of either saline (control animals) or 1% ovalbumin using a deVilbiss Ultraneb nebulizer.
• AHR measurement Eighteen to twenty four hours after challenge, animals are anesthetized with a combination of ketamine (133 mg/kg) and xylazine (24 mg/kg) given intramuscularly, surgically prepared and then mounted in a whole body plethysmograph for lung function measurement. Animals are connected to Ugo-Basile ventilators delivering a tidal volume of 1 mL/lOOg at a rate of 50 breaths/minute via a tracheal cannula. The jugular vein is also cannulated for histamine challenge. A water filled esophageal cannula is placed such that transpulmonary pressure is recordable.
• Transpul- monary pressure is measured as the difference between the tracheal and esophageal cannulas using a differential pressure transducer.
• the volume, airflow, and transpulmonary pressure signals are monitored using a pulmonary analysis system (Buxco XA software) and used to calculate pulmonary resistance (cm ⁇ . 2 0/T ⁇ L/S) and dynamic compliance (mL/cm H 2 O). Airway resistance and dynamic compliance are computed on a breath by breath basis.
• Histamine is administered intravenously and reactivity to increasing concentrations (0.3-20 ⁇ g/kg) assessed.
• ED 50 's are estimated from the area under the curve (AUC) values derived from the individual histamine dose-response curves.
• Plasma and lung drug levels are estimated from the area under the curve (AUC) values derived from the individual histamine dose-response curves.
• Plasma and lung compound levels are determined in satellite groups. Three to four guinea pigs from each of the experimental-drug treatment groups are used for the determination of drug levels. At the indicated time point, (either 2-or 24 hours after dosing), animals are euthanized, and 1 mL blood samples are obtained by cardiac puncture and collected into heparin-coated syringes containing 20 ⁇ L (per 1 mL of blood) of a 5 mM hydralazine solution. Plasma is separated from the cellular component of the blood by centrifugation, and stored at -20°C until assayed.
• Lung samples are dissected free of connective tissues, blotted dry, weighed and stored in 20 mL vials containing 5 mL of a 5 mM hydralazine solution in saline. The frozen plasma samples are then transferred on dry ice to the Pilot PK group for compound levels determination.
• Efficacy of compound I is profiled on Airway Hyperresponsiveness (AHR) to histamine in sensitized guinea pigs, via oral route.
• AHR Airway Hyperresponsiveness
• Compound I has no effect on basal airway resistance or basal dynamic lung compliance.
• Sensitization and single challenge with ovalbumin results in an increase in bronchial reactivity to histamine as denoted by a leftward shift in the dose-response curve of the spasmogen and also by a significant increase in the area under the curve (AUC) for both airway resistance and lung compliance.
• Absolute values represent an increase in airway resistance and a decrease in lung compliance.
• compound I levels are measured in bronchoalveaolar lavage fluid (BAL), lung and plasma, 2 hours after compound dosing (lmg/kg, p.o.). There is an appreciable amount of compound I in target organs such lung and BAL (not taking into account dilution factor for BAL). Plasma compound levels are detected at a much lower level.
• BAL bronchoalveaolar lavage fluid
• compound I levels in both lung and plasma, are also measured 24 hours after dosing. While no plasma could be detected, there is, however, a dose-dependent appreciable amount of compound I detected in guinea pig lung 24 hours after dosing.
• Compound I is also shown to possess a long duration of action with an average ED 50 of 0.4 mg/kg when dosed 24 hours prior to antigen challenge. This long duration of action is in agreement with its extended long exposure.
• the oral data of the compound of the present invention in the guinea pig model of airway hypenesponsiveness clearly shows that the compound exhibits tryptase inhibition activity. Consequently, the compound of the present invention readily has application as a pharmaceutical for treating a wide variety of tryptase related conditions, and naturally, in methods for treating such conditions in a patient.

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